Cell Cycle Checkpoint Regulation Research Articles

Methanolic extract of Citrullus colocynthis suppresses growth and proliferation of breast cancer cells through regulation of cell cycle.

Breast cancer is a major cause of cancer related deaths in women worldwide. Available treatments pose serious limitations such as systemic toxicity, metastasis, tumor recurrence, off-target effects, and drug resistance. In recent years, phytochemicals such as secondary metabolites due to their effective anticancer potential at very low concentration have gained attention. Aim of the study was to evaluate anticancer potential of Citrullus colocynthis and its possible molecular targets on MCF-7, a human breast cancer cell line. Methanolic extract of leaves was prepared and fractionated by solvents (n-hexane, chloroform, ethyl acetate and n-butanol) with increasing polarity. Bioassays and gene expression regulation was conducted to evaluate the anticancer activity, proliferation rate and cell cycle regulation of breast cancer cells treated with extract and its fractions, separately. Results showed a significant anticancer activity of methanolic extract of C. colocynthis and two of its fractions prepared with chloroform and ethyl acetate. Bioassays depicted significant decrease in proliferation and growth potential along with cell cycle arrest of treated cells compared to control untreated cells. Expression regulation of genes further confirmed the cell cycle arrest through significant upregulation of cyclin-CDK inhibitors (p21 and p27) and cell cycle checkpoint regulators (HUS1, RAD1, ATM) followed by downregulation of downstream cell cycle progression genes (Cyclin A, Cyclin E, CDK2). It is concluded that C. colocynthis arrests cell cycle in human breast cancer cells through expression regulation of cyclin-CDK inhibitors and with further research can be proposed for therapeutic interventions.

Design, synthesis, and structure activity relationship (SAR) studies of novel imidazo[1,2-a] pyridine derivatives as Nek2 inhibitors

Never in mitosis (NIMA) related kinase 2 (Nek2) is involved in multiple cellular processes such as cell cycle checkpoint regulation, cell division, DNA damage response and cell apoptosis. Nek2 has been reported to be overexpressed in various tumors and correlated with poor prognosis. Herein, a series of imidazo[1,2-a] pyridines Nek2 inhibitors were designed, synthesized, and their biological activities were investigated. Besides, structure activity relationship analysis of these compounds were performed in the MGC-803 cell. The screening results are promising, and compound 28e shows good proliferation inhibitory activity with an IC50 of 38 nM. The results would be helpful to design and develop more effective Nek2 inhibitors for the treatment of gastric cancer.

An Intimate Alliance of DNA-Damage Response Network with Cell-Cycle Checkpoints Amid Events of Uncontrolled Cellular Proliferation: A Mini- Review.

There is close interdependence between cell survival, cell senescence, events of the cell cycle, apoptosis, malignancy development, and tumor responses to cancer treatment. Intensive studies and elaborate researches have been conducted on the functional aspects of oncogenes, tumor suppressor genes, apoptotic genes, and members guiding cell cycle regulation. These disquisitions have put forward the existence of a highly organized response pathway termed as a DNA-damage response network. The pathways detecting DNA damage and signaling are intensively linked to the events of cell-cycle arrest, cell proliferation, apoptosis, and cell senescence. DNA damage responses are complex systems that incorporate specific "sensor" and "transducer" proteins, for assessment of damage and signal transmission, respectively. These signals are thereafter relayed upon various "effector" proteins involved in different cellular pathways. It may include those governing cell-cycle checkpoints, participating in DNA repair, cell senescence, and apoptosis. This review discusses the role of the tumour suppressor gene, oncogenes, cell cycle checkpoint regulators during DNA damage response and regulation.

Abstract 1329: Identifying low allele frequency somatic variants using the Saphyr System

Abstract Cancer is known to be a dynamic disease with subclones evolving throughout the stages. These progresses create a sample of high heterogeneity, in which high throughput and high sensitivity structural variant calling sensitivity are essential to detect the low frequency events in order to have a thorough understanding of the cancer. Using the Bionano Saphyr® System, a genome imaging platform with a throughput of up to 1.3 Tbp per flowcell, sensitive detection of these low allele frequency structural rearrangements is facilitated by the Bionano data analysis tools, the Rare Variant Pipeline and Variant Annotation Pipeline. Here we compared three tumor-normal pairs of cell lines: the HCC2218, HCC1395 and HCC1954. We first collected about 1.3 Tbp for each of the six tumor and normal cell lines. Each dataset has molecule N50s above 270 kbp. These datasets were input into the Rare Variant Pipeline, where molecules deviating from the reference were locally assembled and structural variants were detected with the local assemblies. Running the Variant Annotation Pipeline then compared the structural variants between the tumor and the control samples and identified variants that are somatic in each pair. With the somatic calls found, we identified various numbers of low allele frequency events in each pair. For example, we observed a low frequency translocation t(2,12) that is unique to the HCC1954 tumor sample. This translocation involves the protein kinase ZAK, a gene known to take part in cell cycle checkpoint regulation (Tosti et al., 2004). In the HCC2218 tumor sample, we found a 381 kbp deletion at AUTS2 (autism susceptibility candidate 2), a gene which has also been associated with acute lymphoblastic leukemia (Denk et al., 2012) and other cancers (Stadler et al., 2012). These events are of low allele frequency and are found to be somatic. In conclusion, cancer samples are heterogeneous and thus, high throughput and sensitive structural variant calling methods are required for a complete examination of the structural mutations that may be present in tumor samples. Bionano has provided an efficient and streamlined data collection platform as well as a smooth and intuitive bioinformatics workflow for expeditious detection of low allele frequency events such as those herein illustrated. References Denk, Dagmar, et al. "PAX5-AUTS2: a recurrent fusion gene in childhood B-cell precursor acute lymphoblastic leukemia." Leukemia research 36.8 (2012): e178-e181. Stadler, Zsofia K., et al. "Rare de novo germline copy-number variation in testicular cancer." The American Journal of Human Genetics 91.2 (2012): 379-383. Tosti, Elena, et al. "The stress kinase MRK contributes to regulation of DNA damage checkpoints through a p38γ-independent pathway." Journal of Biological Chemistry 279.46 (2004): 47652-47660. Citation Format: Joyce Lee, Andy Wing Chun PANG, Caspar Groß, Jakob Admard, Elena Buena-Atienza, Stephan Ossowski, Thomas Anantharaman, Mark Oldakowski, Sven Bocklandt, Alex Hastie. Identifying low allele frequency somatic variants using the Saphyr System [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1329.

Targeting the ABC transporter ABCB5 sensitizes glioblastoma to temozolomide-induced apoptosis through a cell-cycle checkpoint regulation mechanism

Glioblastoma multiforme (GBM) is a malignant brain tumor with a poor prognosis resulting from tumor resistance to anticancer therapy and a high recurrence rate. Compelling evidence suggests that this is driven by subpopulations of cancer stem cells (CSCs) with tumor-initiating potential. ABC subfamily B member 5 (ABCB5) has been identified as a molecular marker for distinct subsets of chemoresistant tumor-initiating cell populations in diverse human malignancies. In the current study, we examined the potential role of ABCB5 in growth and chemoresistance of GBM. We found that ABCB5 is expressed in primary GBM tumors, in which its expression was significantly correlated with the CSC marker protein CD133 and with overall poor survival. Moreover, ABCB5 was also expressed by CD133-positive CSCs in the established human U-87 MG, LN-18, and LN-229 GBM cell lines. Antibody- or shRNA-mediated functional ABCB5 blockade inhibited proliferation and survival of GBM cells and sensitized them to temozolomide (TMZ)-induced apoptosis in vitro Likewise, in in vivo human GBM xenograft experiments with immunodeficient mice, mAb treatment inhibited growth of mutant TP53, WT PTEN LN-229 tumors, and sensitized LN-229 tumors to TMZ therapy. Mechanistically, we demonstrate that ABCB5 blockade inhibits TMZ-induced G2/M arrest and augments TMZ-mediated cell death. Our results identify ABCB5 as a GBM chemoresistance marker and point to the potential utility of targeting ABCB5 to improve current GBM therapies.

Telomerase Biology Associations Offer Keys to Cancer and Aging Therapeutics.

BackgroundAlthough telomerase has potential for age-related disease intervention, the overexpression of telomerase in about 90% of cancers, and in HIV virus reservoirs, cautions against se in anti-aging telomerase therapeutics. While multiple reviews document the canonical function of telomerase for maintenance of telomeres, as well as an increasing numbers of reviews that reveal new non-canonical functions of telomerase, there was no systematic review that focuses on the array of associates of the subunit of Telomerase Reverse transcriptase protein (TERT) as pieces of the puzzle to assemble a picture of the how specific TERT complexes uniquely impact aging and age-related diseases and more can be expected.MethodsA structured search of bibliographic data on TERT complexes was undertaken using databases from the National Center for Biotechnology Information Pubmed with extensive access to biomedical and genomic information in order to obtain a unique documented and cited overview of TERT complexes that may uniquely impact aging and age-related diseases.ResultsThe TERT associations include proper folding, intracellular TERT transport, metabolism, mitochondrial ROS (Reactive Oxygen Species) regulation, inflammation, cell division, cell death, and gene expression, in addition to the well-known telomere maintenance. While increase of cell cycle inhibitors promote aging, in cancer, the cell cycle check-point regulators are ambushed in favor of cell proliferation, while cytoplasmic TERT protects a cell cycle inhibitor in oxidative stress. The oncogene cMyc regulates gene expression for overexpression of TERT, and reduction of cell cycle inhibitors-the perfect storm for cancer promotion. TERT binds with the oncogene RMRP RNA, and TERT-RMRP function can regulate levels of that oncogene RNA, and TERT in a TBN complex can regulate heterochromatin. Telomerase benefit and novel function in neurology and cardiology studies open new anti- aging hope. GV1001, a 16 amino acid peptide of TERT that associates with Heat Shock Proteins (HSP’s), bypasses the cell membrane with remarkable anti disease potential.ConclusionsTERT “associates” are anti-cancer targets for downregulation, but upregulation in antiaging therapy. The overview revealed that unique TERT associations that impact all seven pillars of aging identified by the Trans-NIH Geroscience Initiative that influence aging and urge research for appropriate targeted telomerase supplements/ stimulation, and inclusion in National Institute on Aging Intervention Testing Program. The preference for use of available “smart drugs”, targeted to only cancer, not off-target anti- aging telomerase is implied by the multiplicity of TERT associates functions.

Pulsed low dose-rate irradiation response in isogenic HNSCC cell lines with different radiosensitivity.

BackgroundManagement of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity.Materials and methodsCell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR.ResultsRadioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed.ConclusionsWe demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRS-positive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints.

Deletion of p21 expression accelerates cartilage tissue repair via chondrocyte proliferation

Articular cartilage tissue has a poor healing potential, and when subjected to traumatic damage this tissue undergoes cartilage degeneration and osteoarthritis. The association between the regulation of cell cycle checkpoints and tissue regeneration has been previously investigated, and p21 was initially identified as a potent inhibitor of cell cycle progression. However, the effects of p21 defects on damaged tissue remain controversial. Therefore, the aim of the present study was to evaluate the effects of p21 deficiency on cartilage repair. A mouse model of articular cartilage repair was generated by inducing a patellar groove scratch in 8‑week‑old p21‑knockout (KO) mice and C57Bl/6 wild‑type (WT) mice. Mice were sacrificed at 4and8weeks post‑surgery. The present study also investigated the effect of p21 deficiency on cartilage differentiation in ATDC5 cells invitro. Safranin O staining results indicated that cartilage repair initially occurred in p21 KO mice. In addition, immunohistochemical analysis demonstrated that p21 KO upregulated proliferating cell nuclear antigen and increased cell proliferation. However, typeII collagen and Sox9 expression levels remained unchanged in p21 KO and WT mice. Moreover, it was identified that p21 downregulation did not affect Sox9 and typeII collagen expression levels invitro. Furthermore, p21 deficiency promoted healing of articular cartilage damage, which was associated with cell proliferation invivo, and increased chondrocyte proliferation but not differentiation invitro. Therefore, the present results suggested that p21 does not affect Sox9 or typeII collagen expression levels during cartilage differentiation in the repair of cartilage defects.

Elucidating the role of excision repair cross-complement group 1 in oral epithelial dysplasia and early invasive squamous cell carcinoma: An immunohistochemical study.

Objectives:Oral epithelial dysplasia (OED) is characterized by cellular alterations which have the proclivity of progressing to squamous cell carcinoma. Excision repair cross-complement group 1 (ERCC1) is one of the key proteins involved in nucleotide excision repair (NER) pathway. The expression of ERCC1 has been studied in colorectal, esophageal, ovarian and oral squamous cell carcinoma; but, very few studies have been done to apprehend the expression of ERCC1 in OED and early invasive squamous cell carcinoma (EISCC). The goal of this study is to evaluate the role of ERCC1 in OED and EISCC.Materials and Methods:Histopathologically diagnosed cases of moderate dysplasia (n = 10), severe dysplasia (n = 10) and EISCC (n = 10) were retrieved. 4 μ thick sections were cut from the formalin-fixed paraffin-embedded tissue blocks. The sections were immunohistochemically stained for ERCC1 following standard protocols. The expression of ERCC1 was evaluated semiquantitatively. Statistical analysis was carried out using Fischer's exact t-test.Results:The expression of ERCC1 was found to be strong (+3) in EISCC, moderate (+2) in severe dysplasia and mild (+1) in moderate dysplasia. Thus, the results were statistically significant between the three groups (P < 0.001).Conclusion:Disruption in the mechanisms that regulate cell cycle checkpoints and DNA repair mechanism results in genomic instability; these alterations might contribute to carcinoma. ERCC1 is essential to repair the DNA damage induced by various carcinogens. The present study shows significant difference in the expression of ERCC1 between EISCC and OED, which suggests ERCC1 could be used as one of the predictive markers.

ARID5B Influences Antimetabolite Drug Sensitivity and Prognosis of Acute Lymphoblastic Leukemia.

Treatment outcomes for childhood acute lymphoblastic leukemia (ALL) have improved steadily, but a significant proportion of patients still experience relapse due to drug resistance, which is partly explained by inherited and/or somatic genetic alternations. Recently, we and others have identified genetic variants in the ARID5B gene associated with susceptibility to ALL and also with relapse. In this study, we sought to characterize the molecular pathway by which ARID5B affects antileukemic drug response in patients with ALL. We analyzed association of ARID5B expression in primary human ALL blasts with molecular subtypes and treatment outcome. Subsequent mechanistic studies were performed in ALL cell lines by manipulating ARID5B expression isogenically, in which we evaluated drug sensitivity, metabolism, and molecular signaling events. ARID5B expression varied substantially by ALL subtype, with the highest level being observed in hyperdiploid ALL. Lower ARID5B expression at diagnosis was associated with the risk of ALL relapse, and further reduction was noted at ALL relapse. In isogenic ALL cell models in vitro, ARID5B knockdown led to resistance specific to antimetabolite drugs (i.e., 6-mercaptopurine and methotrexate), without significantly affecting sensitivity to other antileukemic agents. ARID5B downregulation significantly inhibited ALL cell proliferation and caused partial cell-cycle arrest. At the molecular level, the cell-cycle checkpoint regulator p21 (encoded by CDKN1A) was most consistently modulated by ARID5B, plausibly as its direct transcription regulation target. Our data indicate that ARID5B is an important molecular determinant of antimetabolite drug sensitivity in ALL, in part, through p21-mediated effects on cell-cycle progression.

New strategies in ovarian cancer treatment.

Insights from basic science dissecting carcinogenesis in the fallopian tube and ovary have led to a deeper understanding of the origin, molecular characteristics, and types of ovarian cancers. This logically then has led to the development of novel approaches to treat ovarian cancer. Increasingly, novel agents are being developed to target the different growth pathways. The identification of molecular markers associated with different histopathologies has resulted in newer clinical trial designs to capture both clinical and translational endpoints. Unique molecular characteristics in DNA damage and repair pathways and unique cell surface markers have driven new drug development, yielding promise for both patients with platinum-sensitive and platinum-resistant ovarian cancers. Specific examples described include the histology-selective mutations, such as ARID1A in clear cell and endometrioid ovarian cancers; the rationale for using cell cycle checkpoint inhibitors when there already is a p53-mediated loss of cell cycle checkpoint regulation or combinations of agents that will both induce neoantigen formation and unleash immune modulators; and techniques to enhance the therapeutic delivery of known agents. A systematic and thoughtful approach to combining agents in clinical trials is needed so that irrespective of the trial outcomes, the results inform both clinical and translational endpoints.

The atypical cyclin-like protein Spy1 overrides p53-mediated tumour suppression and promotes susceptibility to breast tumourigenesis

BackgroundBreast cancer is the most common cancer to affect women and one of the leading causes of cancer-related deaths. Proper regulation of cell cycle checkpoints plays a critical role in preventing the accumulation of deleterious mutations. Perturbations in the expression or activity of mediators of cell cycle progression or checkpoint activation represent important events that may increase susceptibility to the onset of carcinogenesis. The atypical cyclin-like protein Spy1 was isolated in a screen for novel genes that could bypass the DNA damage response. Clinical data demonstrates that protein levels of Spy1 are significantly elevated in ductal and lobular carcinoma of the breast. We hypothesized that elevated Spy1 would override protective cell cycle checkpoints and support the onset of mammary tumourigenesis.MethodsWe generated a transgenic mouse model driving expression of Spy1 in the mammary epithelium. Mammary development, growth characteristics and susceptibility to tumourigenesis were studied. In vitro studies were conducted to investigate the relationship between Spy1 and p53.ResultsWe found that in the presence of wild-type p53, Spy1 protein is held ‘in check’ via protein degradation, representing a novel endogenous mechanism to ensure protected checkpoint control. Regulation of Spy1 by p53 is at the protein level and is mediated in part by Nedd4. Mutation or abrogation of p53 is sufficient to allow for accumulation of Spy1 levels resulting in mammary hyperplasia. Sustained elevation of Spy1 results in elevated proliferation of the mammary gland and susceptibility to tumourigenesis.ConclusionsThis mouse model demonstrates for the first time that degradation of the cyclin-like protein Spy1 is an essential component of p53-mediated tumour suppression. Targeting cyclin-like protein activity may therefore represent a mechanism of re-sensitizing cells to important cell cycle checkpoints in a therapeutic setting.

Abstract GMM-060: A NOVEL TREATMENT APPROACH FOR TARGETING CYCLIN E OVER-EXPRESSING OVARIAN CANCERS

Abstract Ovarian cancer lacks therapies that yield complete tumor regression resulting in low cure rates. Cyclin E (CCNE1) is an oncogenic driver that is amplified in 25% of high grade serous ovarian cancers (HGSOC). Cyclin E overexpression (CCNE1HIGH) is associated with poor survival and platinum resistance in these cancers. Effective therapies for CCNE1HIGH ovarian cancers yielding complete and durable responses are lacking. Aberrant expression of Cyclin E leads to unscheduled entry into S phase, premature origin firing, nucleotide depletion, and replication fork stress. The imbalances created by this abnormal progression leads to an increased reliance on cell cycle checkpoint regulators, such as WEE1 and ATR, which counter some of the untoward effects of Cyclin E overexpression. WEE1 is a dual specificity kinase that regulates cell cycle progression by inhibiting both CDK2 and CDK1, thereby inhibiting progression from G1 to S and G2 to M phases, respectively. ATR kinase protects the replication fork from collapse thereby inhibiting G2/M progression so DNA can repair. Additionally, mutations in another G1-S phase checkpoint regulator, TP53, may accentuate dependence on WEE1 and ATR by eliminating an alternative checkpoint pathway that limits CCNE1-CDK2 activity. TP53 mutations are also ubiquitous in HGSOC, further implying the promise of WEE1i and ATRi in selective killing CCNE1HIGH tumors as a rational therapeutic strategy. We tested drug effects on survival, colony formation, cell cycle and apoptosis in vitro and in patient-derived xenograft (PDX) models. Induction of Cyclin E expression in immortalized human fallopian tube secretory epithelial cells (FTSEC) upregulates pChk1, a downstream protein of ATR. Combination of WEE1i with ATRi (WEE1i-ATRi) synergistically decreases cell viability and colony formation in CCNE1HIGH HGSOC cells. Selective inhibition of ATR and WEE1 with siRNA was synergistic supporting that ATR and WEE1 are critical to survival. WEE1i-ATRi dramatically increased cell apoptosis, decreased S phase cells and arrested cells at G2/M phase. Combination inhibition of WEE1 and ATR induced gH2AX and phosphorylation of RPA32, indicating increased double strand DNA break and replication stress. Also, combination inhibition of WEE1 and ATR promoted mitotic catastrophe, as indicated by increased pHH3. Finally, combination WEE1-ATRi is tolerable and results in a 4-fold increase in survival compared to standard chemotherapy or monotherapy in a CCNE1 amplified HGSOC PDX model. In addition, sequential treatment is as effective and less toxic as concomitant WEE1i with ATRi warranting further study. Our studies developed a novel combination treatment approach for targeting Cyclin E over-expressing ovarian cancers. Citation Format: Haineng Xu, Yasuto Kinose, Hyoung Kim, Sergey Medvedev, Erin George, Ronny Drapkin, Eric Brown, Gordon Mills, Fiona Simpkins. A NOVEL TREATMENT APPROACH FOR TARGETING CYCLIN E OVER-EXPRESSING OVARIAN CANCERS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-060.

The Prolonged Inhibition of Chk1/Chk2 Kinases Enhances Genetic Instability and Compromises the Efficacy of Chemotherapy Against Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) cells respond to chemotherapy, or more generally to DNA damages, by activating different DNA Damage Response (DDR) pathways. DDR-pathways regulate cell cycle progression and DNA damages repair. Molecular and functional alterations in key DDR-related genes drastically affect the effectiveness of DNA-damaging treatments in cancer cells. For this reasons selective DDR-inhibitors have been developed in order to sensitize cancer cells against conventional chemotherapy. Despite the proven efficacy of DDR-inhibitors in cancer treatment, only few studies have highlighted the biological consequences the prolonged inhibition of DDR-pathways in cancer cells. We hypothesized that the protracted inhibition of the DDR pathways may generate resistant clones characterized by an increased genetic instability. The aim of the study was to evaluate biological consequences of the prolonged inhibition of two crucial DDR-related kinase such as cell cycle checkpoint kinase 1 (Chk1) and 2 (Chk2) in B cells ALL. In particular, we investigated the consequences of Chk1/Chk2 inhibition in term of increase of genetic instability and in term of responsiveness to chemotherapy agents. Starting from B-ALL cell line NALM-6, we generated a resistant model (hereafter referred as N6R-PF8) by treating the parental cells with increasing concentration of PF-00477736 (Chk1/Chk2 inhibitor) for more than a year and increasing the IC50 value of 10-folds. From a molecular point of view, the N6R-PF8 accumulated significant molecular alterations. SNP microarray analysis highlighted different alterations in DDR-related genes and, in particular, in the ATM/CHK2 pathway. Three regions in copy number LOSS (CN=1) containing several genes involved in cell cycle checkpoint regulation (ATM and NPAT) and in the apoptosis (BIRC2, CASP1 and CASP5) were detectable only in NALM-6 parental cell lines and were copy number neutral in the resistant model. Immunoblotting analysis confirmed that in N6R-PF8 cells the ATM/CHK2 and ATR/CHK1 down-stream pathways were significant over-expressed and activated in comparison to the parental cell. Whole exome sequencing analysis showed that the two cell lines were characterized by different mutational profiles and that N6R-PF8 cells harboured significantly more genetic alterations in comparison with NALM-6 cells. Interestingly, crucial genes involved in DNA repair pathway (MLH3, NBN, POLD1 and PMS2) have been found altered only in N6R-PF8 cells. From a functional point of view, the molecular alterations characterizing the N6R-PF8 significantly compromised the cytotoxicity of PF-00477736 and of different DNA damaging agents in comparison to parental cells. Furthermore, the treatment with ATR/ATM inhibitor restored the sensitivity of N6R-PF8 to PF-00477736 and to different chemotherapy agents. In this scenario the level of expression of these two kinases seems to correlate with the sensitivity to DNA damaging agents and to PF-00477736. Finally, we confirmed that the protracted inhibition of crucial DDR-related kinase may increase the overall genetic instability in ALL cells and compromise the efficacy of DNA damaging based therapies. Disclosures Martinelli: Roche: Consultancy; ARIAD: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy.

Modeling of Cisplatin-Induced Signaling Dynamics in Triple-Negative Breast Cancer Cells Reveals Mediators of Sensitivity

SummaryTriple-negative breast cancers (TNBCs) display great diversity in cisplatin sensitivity that cannot be explained solely by cancer-associated DNA repair defects. Differential activation of the DNA damage response (DDR) to cisplatin has been proposed to underlie the observed differential sensitivity, but it has not been investigated systematically. Systems-level analysis—using quantitative time-resolved signaling data and phenotypic responses, in combination with mathematical modeling—identifies that the activation status of cell-cycle checkpoints determines cisplatin sensitivity in TNBC cell lines. Specifically, inactivation of the cell-cycle checkpoint regulator MK2 or G3BP2 sensitizes cisplatin-resistant TNBC cell lines to cisplatin. Dynamic signaling data of five cell cycle-related signals predicts cisplatin sensitivity of TNBC cell lines. We provide a time-resolved map of cisplatin-induced signaling that uncovers determinants of chemo-sensitivity, underscores the impact of cell-cycle checkpoints on cisplatin sensitivity, and offers starting points to optimize treatment efficacy.

Abstract 2046: Egr-1 promotes bone marrow fibrosis in primary myelofibrosis

Abstract Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by JAK-STAT-activated proliferation of leukemia cells and progressive bone marrow fibrosis. However little is known about the bone marrow stromal cell (BMSC) intrinsic factors that work in concert with leukemia stem cells (LSCs) to promote fibrosis of the bone marrow in PMF. In this study we developed an in vitro model to simulate bone marrow fibrosis in PMF by exposing cultured BMSC, HS-5 cells to PMF-patient plasma. We observed that exposure of BMSCs to PMF plasma but not normal serum increased total cellular and mitochondrial reactive oxygen species (ROS), as well as Early growth response 1 (Egr-1) and its target gene expression in BMSCs. Egr-1 is a transcription factor that upregulates fibrosis-associated genes, and is acutely induced by numerous cytokines (e.g. TGF-β, PDGFα) that are elevated in PMF. Utilizing this model, we performed RNA sequencing of HS-5 cells exposed to PMF plasma. We observed that PMF plasma exposure alters pathways responsible for multiple DNA damage responses, G1/s cell cycle checkpoint regulation and protein homeostasis. Since PMF progression is associated with enhanced oxidative stress which induces both DNA damage and Egr-1 expression, we determined the effect of anti-oxidants on the induction Egr-1 and fibrosis associated genes in BMSCs following treatment with PMF plasma. We report that treatment with N-acetyl-cysteine (a ROS scavenger) and mitotempo (a mitochondrial ROS scavenger) abrogated the up-regulation of PMF plasma-induced fibrosis-associated genes in BMSCs. Additionally, knockdown of Egr-1 in BMSCs resulted in the down-regulation of collagen 1A1 and connective tissue growth factor (CTGF) following exposure to PMF plasma. These findings suggest that PMF plasma induces ROS and promotes the upregulation of pro-fibrotic genes in an Egr-1-dependent manner in PMF. Our studies suggest that inhibition of ROS and Egr-1-induced gene transcription could be used as a strategy to alleviate bone marrow fibrosis; a disease complication in PMF for which there are no approved therapies. Citation Format: Rekha M. Rao, Amar Kumar, Pratikkumar Vekaria, Abdulraheem Yacoub, Barry Skikne, Joseph McGuirk. Egr-1 promotes bone marrow fibrosis in primary myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2046.

Abstract 2595: HMGA1 induces the HOXB13 developmental gene to drive tumor progression in androgen receptor negative, castrate-resistant prostate cancer

Abstract While High Mobility Group A1 (HMGA1) chromatin remodeling proteins are frequently dysregulated in diverse cancers, their molecular underpinnings in carcinogenesis remain poorly understood. HMGA1 are small, non-histone proteins that bind to AT-rich regions in DNA, bend chromatin, and recruit transcriptional complexes to modulate gene expression. Prior studies in prostate cancer (PCa) suggest that HMGA1 overexpression associates with higher pathologic grades and increases in HMGA1 protein levels correlate with metastatic potential in rat PCa cells. Overexpression of HMGA1 in human PCa cell lines induces unbalanced chromosomal rearrangements in vitro. Thus, we hypothesized that HMGA1 drives PCa progression through epigenetic reprogramming of transcriptional networks involved in development and chromosomal instability. To test this, we focused on androgen receptor (AR)-negative, castrate-resistant PCa (CRPC) cell lines as tumors with these features are resistant to therapy and associated with metastatic progression and early death. Here, we uncover a novel role for HMGA1 in regulating HOXB13 to drive tumor progression and cancer stem cell properties. We found that silencing HMGA1 in patient-derived metastatic cell lines (PC3-Epi, PC3-EMT, DU145) halts proliferation. Cell morphology changed most dramatically in PC3-EMT cells, transforming spindle-shaped, mesenchymal cells to more cuboidal, epithelial-like cells. Both migration and invasion were disrupted, but primarily in more invasive, mesenchymal cell lines (PC-EMT, DU145). Colony formation and 3D sphere formation were also blocked in all CRPC lines. Immunohistologic analysis in primary tumors revealed that HMGA1 nuclear staining associates with more advanced Gleason scores in PCa. To elucidate transcriptional networks downstream of HMGA1, RNA-seq was performed in PC3-EMT and PC3-Epi cell lines + HMGA1 silencing. Intriguingly, HMGA1 regulates pathways involved in inflammation in the more epithelial PC3-Epi cells, while pathways involved in mitosis, cell cycle progression, DNA damage, and checkpoint regulation predominated in the mesenchymal PC3-EMT cells. Pathways involved in proliferation and development were regulated by HMGA1 in both settings. We focused on HOXB13, a developmental gene linked to cell fate and prostate carcinogenesis. Both HOXB13 and HMGA1 are co-regulated in CRPC cell lines at the gene expression and protein level. HMGA1 occupies at least 1 site within the HOXB13 promoter region by chromatin immunoprecipitation. Strikingly, silencing HOXB13recapitulates HMGA1 phenotypes, impairing proliferation, colony formation, and 3D sphere formation. These findings reveal a novel role for HMGA1 in CRPC progression by dysregulating developmental networks. Together, these results also suggest that targeting the HMGA1-HOXB13 pathway could be effective therapy in CRPC. Citation Format: Lionel Chia, Guangjing Zhu, Mikhail Gorbounov, Lingling Xian, Briyana Chisholm, Mohammad Heydarian, Dorhyun Johng, William B. Isaacs, Karen Reddy, Linda S. Resar. HMGA1 induces the HOXB13 developmental gene to drive tumor progression in androgen receptor negative, castrate-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2595.

PF169 LONG‐TERM INHIBITION OF CHK1/CHK2 KINASES MODIFY ACUTE LYMPHOBLASTIC LEUKEMIA CELL LINE RESPONSE TO DNA DAMAGING AGENTS

Background:Acute lymphoblastic leukemia (ALL) cells respond to chemotherapy, or more generally to DNA damages, by activating different DNA Damage Response (DDR) pathways. DDR‐pathways regulate cell cycle progression and DNA damages repair. Molecular and functional alterations in key DDR‐related genes drastically affect the effectiveness of DNA‐damaging treatments in cancer cells. For this reasons selective DDR‐inhibitors have been developed in order to sensitize cancer cells against conventional chemotherapy. Despite the proven efficacy of DDR‐inhibitors in cancer treatment, only few studies have highlighted the biological consequences the prolonged inhibition of DDR‐pathways in cancer cells.Aims:The aim of the study was to evaluate biological consequences of the prolonged inhibition of two crucial DDR‐related kinase, the cell cycle checkpoint kinase 1 (CHK1) and 2 (CHK2), in B‐ALL cell lines.Methods:The resistant cell lines were obtained from B‐ALL NALM‐6 cell line by prolonged incubation with a CHK1/CHK2 inhibitor, PF‐00477736. Parental cells were progressively incubated with increasing concentration of PF‐00477736 (from 100 nM to 10uM). The functional characterization of resistant cell lines was evaluated in vitro in term of response to different chemotherapy agents (doxorubicin, methotrexate and clofarabine) in term of reduction of cell viability, induction of apoptosis, cell cycle perturbation and activation of DDR pathways. The molecular characterization of resistant cell lines was evaluated in term of Copy Number Alteration (SNP‐array, Affimetrix).Results:Starting from NALM‐6 parental cell lines, we generated two resistant models, N6R‐PF4 and N6R‐PF8. N6R‐PF8 was generated form N6R‐PF4 cells after a prolong incubation with increased PF‐00477736 concentration. In term of reduction of cell viability and induction of apoptosis the resistant cell lines were not only significantly unresponsive to PF‐00477736 (increasing the IC50 up to 8‐fold) but also to different DNA damaging agents in comparison to parental NALM‐6. The cell cycle profile revealed that the resistant cell lines had a significant reduction in the percentage of cells in S phase and a significant increase of cells in G2/M phase. SNP microarray highlighted different alterations in DDR‐related genes and in particular in the ATM/CHK2 pathway. Three regions in copy number LOSS (CN = 1) containing several genes involved in cell cycle checkpoint regulation (ATM and NPAT) and in the apoptosis (BIRC2, BIRC3, CARD17, CASP1, CASP12 and CASP5) were detectable only in NALM‐6 parental cell lines and were copy number neutral in the two resistant models. Immunoblotting analysis confirmed that in the resistant cell lines the ATM/CHK2 down‐stream pathways was significant over‐expressed and activated in comparison to the parental NALM‐6. By chromosome binding analysis we identified that NALM‐6 cells were composed by a predominant clone with ATM locus CN loss and by a sub‐clone (45,X,‐Y,t(5;12)(q33;p13),t(7;19)(q11;p13)) with ATM locus in CN neutral. The treatment with PF‐00477736 progressively selected the sub‐clone, reaching 60% of clones in N6R‐PF4 and 100% of clone in N6R‐PF8.Summary/Conclusion:We showed that the higher protein expression of ATM and CHK2 affected not only the sensitivity to PF‐00477736 but also compromised the efficacy of conventional chemotherapy. In this scenario the level of expression of these two kinases seems to correlate with the sensitivity to DNA damaging agents and to PF‐00477736.

ATM, DNA-PKcs and ATR: shaping development through the regulation of the DNA damage responses

Genomic integrity is critical for normal development, healthy aging and suppressing oncogenic transformation. The DNA damage response (DDR) is a complex network that is activated by DNA structural changes to preserve genome integrity. Situated at the apex of the mammalian DDR are three PI3-kinase-related protein kinases—ATM, DNA-PKcs and ATR. They are activated by different DNA lesions via direct binding to their unique sensor protein complexes (MRE11-RAD50-NBS1 for ATM, Ku70-Ku80/86 for DNA-PKcs and ATRIP-RPA for ATR) and phosphorylate a large number of partially overlapping substrates, including themselves and each other to promote DNA repair and regulate cell cycle checkpoints and tissue homeostasis. This review focuses on mouse models with deletion and point mutations of ATM, DNA-PKcs and ATR, and discusses how their activation mechanism and their kinase activity contribute to their unique, yet interactive roles in DNA repair in general and during tissue-specific development processes and how their deficiency leads to specific physiological and pathophysiological consequences.

Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes.

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies.